Design, Synthesis, and Biological Evaluation of Indole-2-carboxamides as Potential Multi-Target Antiproliferative Agents

A small set of indole-based derivatives, IV and Va–I, was designed and synthesized. Compounds Va–i demonstrated promising antiproliferative activity, with GI50 values ranging from 26 nM to 86 nM compared to erlotinib’s 33 nM. The most potent antiproliferative derivatives—Va, Ve, Vf, Vg, and Vh—were tested for EGFR inhibitory activity. Compound Va demonstrated the highest inhibitory activity against EGFR with an IC50 value of 71 ± 06 nM, which is higher than the reference erlotinib (IC50 = 80 ± 05 nM). Compounds Va, Ve, Vf, Vg, and Vh were further tested for BRAFV600E inhibitory activity. The tested compounds inhibited BRAFV600E with IC50 values ranging from 77 nM to 107 nM compared to erlotinib’s IC50 value of 60 nM. The inhibitory activity of compounds Va, Ve, Vf, Vg, and Vh against VEGFR-2 was also determined. Finally, in silico docking experiments attempted to investigate the binding mode of compounds within the active sites of EGFR, BRAFV600E, and VEGFR-2.


Introduction
Inhibiting oncogenic protein kinases [1][2][3] has been shown to be an effective anticancer strategy [4,5]. As of March 2019, The United States Food and Drug Administration (FDA) has approved a total of 43 small-molecule kinase inhibitors for the treatment of various cancers [6][7][8][9][10]. The majority of approved kinase inhibitors are for receptor tyrosine kinases, the best-validated of which are EGFR (epidermal growth factor receptor) [11] and VEGFR (vascular endothelial growth factor receptor) [12,13]. EGFR amplification or mutation is seen in a variety of cancers, with non-small-cell lung cancer (NSCLC) being the most common [14,15]. VEGFR activation is responsible for tumor angiogenesis/metastasis [16,17] and is associated with a poor prognosis in cancer patients [18,19]. NSCLC is commonly treated with EGFR inhibitors [20,21], as are kidney and thyroid cancers with VEGFR inhibitors [22][23][24].
Protein kinase for serine/threonine Raf (rapid accelerated fibrosarcoma) [25,26], which includes A-Raf, B-Raf, and C-Raf, is a key player in the Ras/Raf/MEK/ERK (MAPK, mitogen-activated protein kinase) signaling pathway [27,28]. Via this pathway, growth signals from cell surface receptors (e.g., EGFR and VEGFR) to the nucleus promote cell proliferation, differentiation, and survival. B-Raf is the most frequently mutated Raf Signals from receptor tyrosine kinases (e.g., EGFR and VEGFR) can also be blocked by downstream Raf inhibition. Resistance to current B-Raf V600E therapy, on the other hand, has been linked to EGFR signaling pathways [35,36] or VEGF-A upregulation [37,38]. The combination of the EGFR antibody cetuximab and drug 1 shows clinical benefits in patients with refractory B-Raf V600E metastatic colorectal cancer [39][40][41]. In vivo, the combination of the B-Raf V600E inhibitor PLX4720 and the VEGF antibody bevacizumab has synergistic effects [42,43]. These findings suggested that a small-molecule Raf inhibitor with EGFR/VEGFR inhibitory activity could be beneficial for cancers that are refractory to other treatments. Ding and colleagues [44] reported a new class of dual B-Raf/EGFR inhibitors in what became a leading study. The improved compound is effective against melanoma and/or colorectal cancers that are resistant to 1.
The indole skeleton, which is found in many active substances and natural products, is one of the most well-known structures with impressive anticancer activity [45][46][47]. Many indole compounds have been found to be effective anticancer medicines, with some even being utilized in clinics [31,[48][49][50]. A substantial number of indole-based compounds with TK inhibitory action were also discovered in the literature study [51][52][53].
Song et al. developed a set of indole derivatives that act as dual EGFR/VEGFR-2 inhibitors [54]. Compound 3 (Figure 1) inhibited EGFR and VEGFR-2 simultaneously, with IC50 values of 18 and 45 nM, respectively. Compound 4 ( Figure 1) was also reported to be a dual EGFR/VEGFR-2 inhibitor, with a stronger effect against EGFR than 3, indicating the importance of the morpholino moiety in EGFR inhibitory activity. Osimertinib (5, Figure 1) is an EGFR TKI that is approximately 200 times more selective for the mutant protein than for wild-type EGFR [53]. Osimertinib was approved by the Signals from receptor tyrosine kinases (e.g., EGFR and VEGFR) can also be blocked by downstream Raf inhibition. Resistance to current B-Raf V600E therapy, on the other hand, has been linked to EGFR signaling pathways [35,36] or VEGF-A upregulation [37,38]. The combination of the EGFR antibody cetuximab and drug 1 shows clinical benefits in patients with refractory B-Raf V600E metastatic colorectal cancer [39][40][41]. In vivo, the combination of the B-Raf V600E inhibitor PLX4720 and the VEGF antibody bevacizumab has synergistic effects [42,43]. These findings suggested that a small-molecule Raf inhibitor with EGFR/VEGFR inhibitory activity could be beneficial for cancers that are refractory to other treatments. Ding and colleagues [44] reported a new class of dual B-Raf/EGFR inhibitors in what became a leading study. The improved compound is effective against melanoma and/or colorectal cancers that are resistant to 1.
The indole skeleton, which is found in many active substances and natural products, is one of the most well-known structures with impressive anticancer activity [45][46][47]. Many indole compounds have been found to be effective anticancer medicines, with some even being utilized in clinics [31,[48][49][50]. A substantial number of indole-based compounds with TK inhibitory action were also discovered in the literature study [51][52][53].
Song et al. developed a set of indole derivatives that act as dual EGFR/VEGFR-2 inhibitors [54]. Compound 3 (Figure 1) inhibited EGFR and VEGFR-2 simultaneously, with IC 50 values of 18 and 45 nM, respectively. Compound 4 ( Figure 1) was also reported to be a dual EGFR/VEGFR-2 inhibitor, with a stronger effect against EGFR than 3, indicating the importance of the morpholino moiety in EGFR inhibitory activity. Osimertinib (5, Figure 1) is an EGFR TKI that is approximately 200 times more selective for the mutant protein than for wild-type EGFR [53]. Osimertinib was approved by the FDA in 2015 to treat EGFR T790M -positive NSCLC due to its selectivity and activity [53,55,56].
Recently, we reported on some indole derivatives that exhibited promising antiproliferative activity as dual or multikinase inhibitors [31,48,[57][58][59][60][61]. Some of these compounds, targeted kinases, and their IC50 values are shown in Figure 2. Motivated by the aforementioned data on the promising antiproliferative action of indole-based structures and as part of our ongoing effort to find dual or multi-targeted kinase agents, in this paper, we present the synthesis and antiproliferative activity of a small set of indole-based derivatives: IV and Va-i ( Figure 3).  Motivated by the aforementioned data on the promising antiproliferative action of indole-based structures and as part of our ongoing effort to find dual or multi-targeted kinase agents, in this paper, we present the synthesis and antiproliferative activity of a small set of indole-based derivatives: IV and Va-i ( Figure 3). We tested the antiproliferative activity of the newly synthesized compounds against four different human cancer cell lines. The most promising derivatives were further investigated for multi-kinase inhibitory effects against EGFR, VEGFR-2, and BRAF V600E . In addition, caspase and apoptotic assay pathways were assessed. Finally, molecular docking studies of the most active compounds within the active sites of targeted kinases were performed to determine the binding modes of the evaluated compounds.

Chemistry
Scheme 1 depicts the synthesis of compounds III, IV, and Va-i. In the presence of catalytic amounts of sulfamic acid, 2-phenyl indole (I) and β-nitrostyrene (II) were reacted in refluxing methanol to produce 3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole (III), which was then purified via flash chromatography on silica gel using EtOAc/hexanes (1:4) to give the desired product as an oil, which was solidified by being dissolved in DCM followed by addition of hexane. Then, compound III was reduced in diethyl ether (Et2O) under a nitrogen atmosphere using lithium aluminum hydride (LAH) to yield the desired product IV as a white solid. The structure of compound IV was confirmed using 1 H NMR, 13 C NMR, and HRESI-MS spectroscopy. The presence of characteristic signals of the ethanamine group (CHCH2NH2) was revealed in the 1 H NMR spectrum of IV as doublet of doublet signal (1H) at δ 4.42 of CHCH2 (J = 9.7, 6.2 Hz), doublet of doublet signal (1H) at δ 3.58 of CHCH2a (J = 12.6, 9.8 Hz), doublet of doublet signal (1H) at δ 3.44 of CHCH2b (J = 12.7, 6.2 Hz), and singlet signal (2H) at δ 1.34 of the NH2 group, which was confirmed by the presence of two carbon signals at δ 46.58 and 46.55 in the 13 C NMR spectrum of IV. Our HRESI-MS analysis of IV revealed the presence of a peak at m/z 313.1700, calculated for [M+H] + C22H21N2: 313.1699. We tested the antiproliferative activity of the newly synthesized compounds against four different human cancer cell lines. The most promising derivatives were further investigated for multi-kinase inhibitory effects against EGFR, VEGFR-2, and BRAF V600E . In addition, caspase and apoptotic assay pathways were assessed. Finally, molecular docking studies of the most active compounds within the active sites of targeted kinases were performed to determine the binding modes of the evaluated compounds.

Chemistry
Scheme 1 depicts the synthesis of compounds III, IV, and Va-i. In the presence of catalytic amounts of sulfamic acid, 2-phenyl indole (I) and β-nitrostyrene (II) were reacted in refluxing methanol to produce 3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole (III), which was then purified via flash chromatography on silica gel using EtOAc/hexanes (1:4) to give the desired product as an oil, which was solidified by being dissolved in DCM followed by addition of hexane. Then, compound III was reduced in diethyl ether (Et 2 O) under a nitrogen atmosphere using lithium aluminum hydride (LAH) to yield the desired product IV as a white solid. The structure of compound IV was confirmed using 1 H NMR, 13 C NMR, and HRESI-MS spectroscopy. The presence of characteristic signals of the ethanamine group (CHCH 2 NH 2 ) was revealed in the 1 H NMR spectrum of IV as doublet of doublet signal (1H) at δ 4.42 of CHCH 2 (J = 9.7, 6.2 Hz), doublet of doublet signal (1H) at δ 3.58 of CHCH 2 a (J = 12.6, 9.8 Hz), doublet of doublet signal (1H) at δ 3.44 of CHCH 2 b (J = 12.7, 6.2 Hz), and singlet signal (2H) at δ 1.34 of the NH 2 group, which was confirmed by the presence of two carbon signals at δ 46.58 and 46.55 in the 13 C NMR spectrum of IV. Our HRESI-MS analysis of IV revealed the presence of a peak at m/z 313.1700, calculated for [M+H]  The desired indole-2-carboxamides Va-i were obtained by coupling ethanamine IV with appropriate indole-2-carboxylic acids (1)(2)(3)(4)(5) in dichloromethane (DCM) using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and diisopropyl ethylamine (DIPEA). Various spectroscopic methods of analysis were used to confirm the structures of compounds Va-i. As an example, the 1 H NMR spectrum of compound Vg (R 1 = methoxyvinyl, R 2 = Cl) revealed the presence of three signals corresponding to NH groups: a singlet signal at δ 9.79 ppm of indole NH, a singlet signal at δ 8.34 ppm of 2-phenylindole NH, and a triplet signal at δ 6.54 ppm of amidic NH. Moreover, the spectrum revealed methoxyvinyl group characteristic signals in the form of two doublets of 1H each δ 6.59 ppm and δ 5.15 ppm corresponding to CH=CHOCH 3 and CH=CHOCH 3 , respectively, and singlet signal of 3H at δ 3.15 ppm of OCH 3 . Also, the Vg spectrum was characterized by the presence of signals corresponding to CHCH 2 group, in the form of The desired indole-2-carboxamides Va-i were obtained by coupling ethanamine IV with appropriate indole-2-carboxylic acids (1)(2)(3)(4)(5) in dichloromethane (DCM) using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and diisopropyl ethylamine (DIPEA). Various spectroscopic methods of analysis were used to confirm the structures of compounds Va-i. As an example, the 1 H NMR spectrum of compound Vg (R1 = methoxyvinyl, R2 = Cl) revealed the presence of three signals corresponding to NH groups: a singlet signal at δ 9.79 ppm of indole NH, a singlet signal at δ 8.34 ppm of 2-phenylindole NH, and a triplet signal at δ 6.54 ppm of amidic NH. Moreover, the spectrum revealed methoxyvinyl group characteristic signals in the form of two doublets of 1H each δ 6.59 ppm and δ 5.15 ppm corresponding to CH=CHOCH3 and CH=CHOCH3, respectively, and singlet signal of 3H at δ 3.15 ppm of OCH3. Also, the Vg spectrum was characterized by the presence of signals corresponding to CHCH2 group, in the form of

Assay for Cell Viability
To assess the viability of compounds IV and Va-i, the human mammary gland epithelial (MCF-10A) cell line was exploited [62][63][64]. Compounds IV and Va-i were cultured for four days on MCF-10A cells before being evaluated for vitality using the MTT assay. According to Table 1, none of the compounds examined displayed cytotoxic actions, and the cell viability for the compounds tested at 50 µM was greater than 89%.

Assay for Cell Viability
To assess the viability of compounds IV and Va-i, the human mammary gland epithelial (MCF-10A) cell line was exploited [62][63][64]. Compounds IV and Va-i were cultured for four days on MCF-10A cells before being evaluated for vitality using the MTT assay. According to Table 1, none of the compounds examined displayed cytotoxic actions, and the cell viability for the compounds tested at 50 µM was greater than 89%.

Antiproliferative Assay
Using the MTT assay [59,65] and erlotinib as the reference drug, the antiproliferative activity of Va-i was assessed against four human cancer cell lines: a pancreatic cancer (Panc-1) cell line, breast cancer (MCF-7) cell line, colon cancer (HT-29) cell line, and human epithelial (A-549) cancer cell line. The median inhibitory concentration (IC 50 ) is shown in Table 1.
Compound IV (the amine derivatives) was the least potent of all the synthesized compounds, with a GI 50 value of 104 nM against the four cancer cell lines tested, approximately three-fold less potent than the reference erlotinib, which has a GI 50 value of 33 nM. In contrast to compound IV, compounds Va-o demonstrated promising antiproliferative activity, with GI 50 values ranging from 26 nM to 86 nM against the cancer cell lines tested compared to erlotinib's 33 nM. In all cases, compounds Va-o were more potent than compound IV, indicating the importance of the second indole or benzofuran moiety for activity.  --: Not Determined

EGFR Inhibitory Assay
The antiproliferative compounds with the highest potency (Va, Ve, Vf, Vg, and Vh) were further examined for their suppressive action against EGFR as a probable target for their antiproliferative activity [59,66]. Table 2 displays the results as IC50 values. This test's results are comparable with those of the antiproliferative assay, with compound Va (R1 = H, R2 = Cl, X = NH) displaying the strongest inhibitory activity against EGFR (IC50 = 71±06 nM), which is greater than the reference erlotinib (IC50 = 80±05 nM).
These results indicate that EGFR may be a potential target for compounds Va, Vg, and Vh, which may require additional structural modifications in the future to optimize their effects. Compounds Va and Ve-h were the most effective antiproliferative agents, with GI 50 ranging from 26 nM to 48 nM. Compound Va (R 1 = H, R 2 = Cl, X = NH) was the most potent derivative, with a GI 50 value of 26 nM, being 1.3-fold more potent than the reference erlotinib. Compound Va outperformed erlotinib against all cancer cell lines tested.
Compound Vg (R 1 = CH=CH-O-CH 3 , R 2 = Cl, X = NH) ranked second in terms of antiproliferative action with a GI 50 value of 31 nM, and it was comparable to the reference erlotinib but more potent against the breast cancer cell line (MCF-7) ( Table 1). Compound Vh (R 1 = CH 2 -O-CH 2 CH 3 , R 2 = Cl, X = NH) revealed promising antiproliferative action with a GI 50 value of 37 nM against the four cancer cell lines tested, which is 1.4-fold less potent than compound Va.
Compounds Ve (R1 = CH 2 OH, R 2 = Cl, X = NH) and Vf (R 1 = Ph, R 2 = Cl, X = NH) demonstrated promising antiproliferative activities, with GI 50 values of 44 nM and 48 nM, respectively, which were 1.3-fold and 1.45-fold less potent than the reference erlotinib. These findings showed that the type and nature of the substituent on the third position of the indole/benzofuran moiety is required for activity and that it increased in the order H > methoxyvinyl > ethoxymethyl > hydroxymethyl > phenyl.
Compound Vc (R 1 = CH 2 CH 3 , R 2 = Cl, X = NH) demonstrated good antiproliferative activity against the four cancer cell lines tested, with a GI 50 value of 56 nM, which is 2.2-fold less potent than Va (R 1 = H, R 2 = Cl, X = NH). Furthermore, compound Vb (R 1 = CH 3 , R 2 = Cl, X = NH) showed an effect that was comparable to that of the 3-ethyl derivative, Vc, with a GI 50 of 59 nM, which is also less potent than Va, confirming the importance of the 3-substitution on the second indole moiety. The substitution of a bromine atom for the chlorine atom in compound Vd (R 1 = CH 2 CH 3 , R 2 = Br, X = NH) resulted in a significant decrease in the antiproliferative activity of compound Vd, which had a GI 50 value of 66 nM (1.2-fold less potent than the chloro derivative Vc), indicating that the chlorine atom is more tolerated for the antiproliferative action than the bromine one.
Finally, the benzofuran derivative Vi (R 1 = CH 2 CH 3 , R 2 = Cl, X = O) exhibited the least potent activity among Va-o derivatives, with a GI 50 value of 86 nM, which is 1.5-fold less potent than the indole derivative Vd (R 1 = CH 2 CH 3 , R 2 = Cl, X = NH), indicating the importance of the indole NH group in antiproliferative action.

EGFR Inhibitory Assay
The antiproliferative compounds with the highest potency (Va, Ve, Vf, Vg, and Vh) were further examined for their suppressive action against EGFR as a probable target for their antiproliferative activity [59,66]. Table 2 displays the results as IC 50 values. This test's results are comparable with those of the antiproliferative assay, with compound Va (R 1 = H, R 2 = Cl, X = NH) displaying the strongest inhibitory activity against EGFR (IC 50 = 71 ± 06 nM), which is greater than the reference erlotinib (IC 50 = 80 ± 05 nM). With an IC 50 value of 79 ± 06 nM, compound Vg (R 1 = CH=CH-O-CH 3 , R 2 = Cl, X = NH) is the second most active compound, followed by compound Vh (R 1 = CH 2 -O-CH 2 CH 3 , R 2 = Cl, X = NH), which has an IC 50 value of 85 ± 08 nM. Compounds Ve and Vf showed moderate EGFR inhibitory activity with IC 50 values of 94 ± 07 nM and 103 ± 08 nM, respectively.
These results indicate that EGFR may be a potential target for compounds Va, Vg, and Vh, which may require additional structural modifications in the future to optimize their effects.

BRAF V600E Inhibitory Assay
Compounds Va, Ve, Vf, Vg, and Vh were further tested for BRAF V600E inhibitory activity, and the results are shown in Table 2 as IC 50 values [31,67]. The results showed that the tested compounds inhibited BRAF V600E with IC 50 values ranging from 77 nM to 107 nM; erlotinib's IC 50 value was 60 nM.
Compound Va (R 1 = H, R 2 = Cl, X = NH), which was the most potent derivative in both antiproliferative and EGFR inhibitory assays, was also the most potent derivative BRAF V600E inhibitor, showing an IC 50 value of 67 ± 5 nM, which was less potent than erlotinib (IC 50 = 60 ± 5 nM). Vg (R 1 = CH=CH-O-CH 3 , R 2 = Cl, X = NH) and Vh (R 1 = CH 2 -O-CH 2 CH 3 , R 2 = Cl, X = NH) ranked second and third as anti-BRAF V600E compounds, with IC 50 values of 83 ± 6 nM and 89 ± 7 nM, respectively. These findings show that compounds Va, Vg, and Vh have potent antiproliferative activity and may act as dual EGFR and BRAF V600E inhibitors.

VEGFR-2 Inhibitory Assay
The upregulation of many kinases in endothelial cells showed a critical involvement in cancer angiogenesis and vasculogenesis [68,69]. VEGFR-2 is one of these protein kinases that are involved in endothelial cell survival/proliferation and cancer development [70,71]. The inhibitory activity of compounds Va, Ve, Vf, Vg, and Vh against VEGFR-2 was determined utilizing kinase-glo-luminescent kinase assays with sorafenib as the reference drug [63,72]. Table 2 presents the results.
In general, the investigated compounds showed good VEGFR-2 inhibitory activity, with IC 50 values ranging from 1.10 nM to 3.25 nM, whereas the reference sorafenib had an IC 50 value of 0.17 nM. Compounds Ve (R 1 = CH 2 OH, R 2 = Cl, X = NH) and Vg (R 1 = CH=CH-O-CH 3 , R 2 = Cl, X = NH) revealed the most potent VEGFR-2 inhibitory effects, with IC 50 values of 1.10 nM and 1.60 nM, respectively, which were six-fold less potent than the reference sorafenib. The most potent derivative in the antiproliferative, EGFR, and BRAF V600E inhibitory assays, compound Va (R 1 = H, R 2 = Cl, X = NH), had a promising inhibitory effect on VEGFR-2 with an IC 50 value of 2.15 ± 0.20 nM. Finally, compound Vg (R 1 = CH=CH-O-CH 3 , R 2 = Cl, X = NH) showed excellent VEGFR-2 inhibitory efficacy with an IC 50 value of 3.25 nM, although this was two-fold less potent than compound Ve.
The antiproliferative, EGFR, BRAF V600E , and VEGFR-2 results showed that compounds Va, Ve, Vg, and Vh can display substantial antiproliferative effects and that they may be able to operate as multi-kinase inhibitors, making them viable lead compounds for additional structural modifications.

Apoptotic Marker Assays
The development of innovative apoptosis-targeting drugs has become crucial for therapeutic application, as apoptotic abnormalities in cancer cells are the most significant barrier to anticancer therapy efficacy [73][74][75][76]. To reveal their proapoptotic potential, compounds Va, Ve, Vg, and Vh were investigated for their ability to trigger the apoptosis cascade.

Caspase 3 Assay
Caspases play a crucial function in the induction and completion of apoptosis. Caspase-3 is an essential caspase that cleaves different proteins in cells, resulting in apoptosis [77][78][79]. The most effective derivatives, compounds Va, Ve, Vg, and Vh, were evaluated as caspase-3 activators against a human epithelial cancer cell line (A-549) [73], and the findings are reported in Table 3. The results showed that the studied compounds Va, Ve, Vg, and Vh had good caspase-3 overexpression levels, ranging from 460 ± 4 up to 726 ± 6 pg/mL in comparison to untreated control cells, which had a caspase-3 level of 66 pg/mL. Compounds Va and Vg displayed outstanding caspase-3 protein overexpression levels of 726 ± 6 and 528 ± 5 pg/mL, respectively. They elevated the protein caspase-3 in the human epithelial cancer cell line (A-549) by roughly 11 and 8 times more than the untreated control cells, and they were even more active than the reference doxorubicin (505 ± 4.0 pg/mL). These findings suggest that the compounds tested operate as caspase-3 activators and can thus be classified as apoptotic inducers.
2.3.2. Caspase-8, Bax, and Bcl-2 Level Assays As indicated in Table 3, compounds Va and Vg were investigated further for their effect on caspase-8, Bax, and Bacl-2 levels against the human epithelial (A-549) cancer cell line using doxorubicin as a control. The results showed that, compared to doxorubicin, all of the tested compounds significantly elevated caspase-8 and Bax levels.
Finally, versus doxorubicin, Va and Vg elicited the equipotent downregulation of anti-apoptotic Bcl-2 protein levels in the A-549 cell line (Table 3).

Molecular Modeling
A computational docking study was performed for compounds IV and Va-i to investigate their binding interactions with the epidermal growth factor receptor tyrosine kinase EGFR. Molecular Operating Environment (MOE) software [80,81] was used along with the crystal structure of the EGFR in complex with erlotinib (PDB: 1M17) [59,82]. All minimizations were completed using the force field (OPLS-AA) as well as Born solvation. The protein structure was protonated and corrected prior to the docking experiment.
The docking protocol was validated through redocking the co-crystallized erlotinib within the EGFR active site as the S score achieved by the docked ligand was -10.70 kcal/mol with RMSD value of 1.48 Å (Figure S1), Table 4. Based on the docking score analysis, the most effective antiproliferative compounds, Va, Ve, Vf, Vg, and Vh, exhibited the highest negative values, ranging from −9.89 to −10.52 kcal/mol compared with erlotinib's score of −10.70 kcal/mol. However, the least potent amine derivative, IV, had the lowest docking score (−7.79 kcal/mol) amongst all compounds. After inspecting the ligand protein complexes, it was revealed that compound IV missed essential binding interactions within the large binding site ( Figure 4A,B). The ligand 2-phenylindole scaffold bound deeply into the hydrophobic pocket forming stacking with Phe699 and pi-H interaction with Val702 (3.91 Å) in a way that was analogous to the erlotinib phenyl acetylene moiety. Also, the ligand amino group forms ionic interactions with Asp831 and Glu738 (3.93 and 3.65 Å, respectively) and donates H-bond interactions to Thr830 and Met742 with 2.79 and 4.19 Å, respectively. However, the ligand neither interacts with the key amino acid Met769 nor forms interactions at the gate of binding site. The introduction of a 5-haloindole moiety in compounds Va-h via amide linkage improved the ligand binding profile within the protein active site. The ligand 5-haloindole moiety, instead, inserted deeply into the hydrophobic pocket forming stacking with Phe699 and pi-H interaction with Leu820. In addition, the 5-haloindole NH donates H-bond interactions to Asp831 in compounds Va-h, with the exception of compound Ve, where the 3-hydroxymethylene group donates similar H-bond interactions to Asp831 with 3.09 Å. The latter interaction was missed in compound Vi, with the 5-halobenzofuran moiety indicating the significance of the indole NH moiety for optimally fitting the ligand within the active site. Moreover, the protein pocket accommodates both chlorine and bromine atoms at the fifth position of the indole moiety as the ligand forms additional halogen bond interactions with Leu764 and/or Thr766 at the pocket hinge. Interestingly, the substitution pattern at the third position of the 5-haloindole moiety has a significant impact on the VDW interaction surface of protein. The unsubstituted compound Va with R 1 = H and compound Vg with planar group R 1 = CH=CH-OCH 3 showed the best fitting within the protein interaction surface. In contrast, with bulkier groups (R 1 = CH 3 , CH 2 CH 3 , CH 2 CH 3 , CH 2 OH, Ph, CH 2 OCH 2 CH 3 , respectively), compounds Vb, Vc, Vd, Ve, Vf, and Vh projected out of the protein interaction surface, destabilizing their overall binding complexes. Finally, the ligand 2-phenyl indole moiety stacked with Phe771 past the erlotinib ether linkages forming pi-H interactions with Leu694 and Gly772 in addition to other hydrophobic interactions with surrounding residues Gly772, Lys721, Leu694, Asp831, and Val702 at the gate of the binding site. (Figure 4C-F). Also, the 2-phenyl indole NH moiety in compounds Vb and Vf donates H-bond interactions to Asp776 with 2.99 and 2.90 Å, respectively. Leu694, Leu820, Val702, Gly722, Thr766, Pro770, Glu780, His781 -------------------- Leu820, Val702, Gly722, Thr766, Leu694, Asp776, Glu780 Leu820 (pi-H) Glu780, Leu694, Leu820, Val702, Gly722, Thr766, Asp776, Leu694, Leu820, Val702, Gly722, Thr766, Asp776, Glu780 Val702 (pi-H) Leu694, Leu820, Val702, Gly722, Thr766, Asp776, Glu780 Thr766, Asp776, Glu780, Leu694, Leu820, Val702, Gly722 Gly695, Val702 (pi-H)  The foremost active antiproliferative compounds, Va, Ve, Vf, Vg, and Vh, were docked in silico to study their binding modes within the active site of BRAF V600E using the crystal structure of the BRAF V600E in complex with Vemurafenib (PDB: 3OG7) [83,84]. The docking results within the protein binding site were validated by redocking the cocrystallized vemurafenib showing an S score of −11.78 kcal/mol with a RMSD value of 0.96 Å ( Figure S2) (Table 5). Our analysis of the docking scores of the examined compounds The foremost active antiproliferative compounds, Va, Ve, Vf, Vg, and Vh, were docked in silico to study their binding modes within the active site of BRAF V600E using the crystal structure of the BRAF V600E in complex with Vemurafenib (PDB: 3OG7) [83,84]. The docking results within the protein binding site were validated by redocking the cocrystallized vemurafenib showing an S score of −11.78 kcal/mol with a RMSD value of 0.96 Å ( Figure S2) (Table 5). Our analysis of the docking scores of the examined compounds showed that compounds Va, Vg, and Vh exhibited better scores than compounds Ve and Vf. After investigating the binding modes, it was revealed that the most potent compound Va (R 1 = H, R 2 = Cl, X = NH) extended snugly within the protein active site ( Figure 5A,B). The compound probes the space of the active site where the ligand 5-chloroindole moiety stacks between Phe583 and Trp531 within the hydrophobic pocket forming hydrophobic interactions with Val471, Trp531, Phe583, Cys532, Ile463, and Thr592. Also, the 5-chloroindole moiety forms pi-H interactions with Val471 and/or Ile527 in compounds Vf, Vg, and Vh. In addition, the 5-chloro indole NH group donates H-bond interactions to the key amino acid residue Thr529 with (2.79 Å). Moreover, the chloro group was close to the key amino acid residue Cys532 at the site gate. Also, the ligand amide NH moiety forms weak H-bond interactions with the key amino acid Lys483. However, the unsubstituted derivative Va missed essential interactions with the amino acid residues Gln530, Cys532, Asp594, Gly596 compared with the co-crystallized ligand, Vemurafenib, at the binding site. The results of the docking simulations against EGFR explained the antiproliferative effects of compounds IV and Va-i relative to their binding affinity within the active site. Further simulations against BRAF V600E suggest that compounds Va, Vg, and Vh might act as dual EGFR and BRAF V600E kinase inhibitors.  Moreover, the most potent antiproliferative compounds (Va, Ve, Vf, Vg, and Vh) were docked against vascular endothelial growth factor VEGFR-2 in order to investigate their potential multi-kinase inhibition, Figure 6. The crystal structure of VEGFR-2 in complex with sorafenib (PDB: 4ASD) [63] was used in the current study. The docking experiment was validated by redocking the co-crystallized ligand that showed an S score of -10.73 kcal/mol with a RMSD value of 0.46 Å, ( Figure S3). All docked compounds showed good scores ranging from −8.18 to −9.77 kcal/mol compared with the co-crystallized ligand ( Table 6). After examining the docked complexes, it was revealed that the compounds exhibited good fitting within the binding pocket. They showed comparable binding modes within the active site. At the hydrophobic gate of the binding site, the ligand 5-chloroindole scaffold stacked with Phe1047 and became surrounded by amino acid residues Phe918 and Phe921. On the opposite end of binding site, the 2-phenylindole moiety forms stacking with His1026 while making pi-H interactions with Cys1045 in the most potent compounds (Ve and Vg). However, the latter amino acid residue forms H-pi interactions with the 5-chloroindole moiety in compound Vf, Figure 6. Despite missing an interaction with key amino acid Cys919, the ligand amide linkage donates a H-bond to Glu885 while accepting a H-bond from Asp1046 in compounds Va, Vf, and Vh. However, in the case of the most potent compound, Ve, the ligand hydroxy methylene and the amide NH donate H-bond interactions to Glu885 and Asp1046 with 2.64 and 3.04 Å, respectively ( Figure 3A,B). Compared with compound Ve, the amide carbonyl group of compound Vg accepts two H-bond interactions from Asp1046 and Cys1045 and misses an interaction with the key amino acid Glu885 due to its ether linkage ( Figure 3C,D). The results of the docking simulations predicted the binding modes of the most active antiproliferative compounds and confirmed their potential multi-kinase inhibitory effects. showed that compounds Va, Vg, and Vh exhibited better scores than compounds Ve and Vf. After investigating the binding modes, it was revealed that the most potent compound Va (R1 = H, R2 = Cl, X = NH) extended snugly within the protein active site ( Figure 5A,B). The compound probes the space of the active site where the ligand 5-chloro-indole moiety stacks between Phe583 and Trp531 within the hydrophobic pocket forming hydrophobic interactions with Val471, Trp531, Phe583, Cys532, Ile463, and Thr592. Also, the 5chloroindole moiety forms pi-H interactions with Val471 and/or Ile527 in compounds Vf, Vg, and Vh. In addition, the 5-chloro indole NH group donates H-bond interactions to the key amino acid residue Thr529 with (2.79 Å). Moreover, the chloro group was close to the key amino acid residue Cys532 at the site gate. Also, the ligand amide NH moiety forms weak H-bond interactions with the key amino acid Lys483. However, the unsubstituted derivative Va missed essential interactions with the amino acid residues Gln530, Cys532, Asp594, Gly596 compared with the co-crystallized ligand, Vemurafenib, at the binding site. The results of the docking simulations against EGFR explained the antiproliferative effects of compounds IV and Va-i relative to their binding affinity within the active site. Further simulations against BRAF V600E suggest that compounds Va, Vg, and Vh might act as dual EGFR and BRAF V600E kinase inhibitors.

In Silico ADME/Pharmacokinetics Studies
In silico ADME studies were performed for compounds IV and Va-i using SwissADME [85,86] as well as ADMET lab tools [87,88]. The compounds' SMILES (Simplified Molecule Input Line Entry Specification), obtained by using ChemDraw software, were entered as a list. The obtained pharmacokinetic data (Table 7) revealed that only compounds IV and Va are likely to be orally active as they obey Lipinski's rules of five with zero or one violation, respectively. All tested compounds are expected to be a Pgp non-substrate. They are considered to be poorly absorbed by the intestine, except for compound IV. They are able to cross BBB with a probability ranging from 0.7 to 0.9. Most compounds exhibited limited permeability as indicated by logP values in the range of 4.25-7.59, except for compound IV. Concerning CYP inhibition, all compounds were predicted as inhibitors with a probability higher than 0.5, as shown in Table 7. The results predict that compounds IV and Va-i could exhibit acceptable pharmacokinetic and ADME properties (Tables 7 and 8).

In Silico ADME/Pharmacokinetics Studies
In silico ADME studies were performed for compounds IV and Va-i using Swis-sADME [85,86] as well as ADMET lab tools [87,88]. The compounds' SMILES (Simplified Molecule Input Line Entry Specification), obtained by using ChemDraw software, were entered as a list. The obtained pharmacokinetic data (Table 7) revealed that only compounds IV and Va are likely to be orally active as they obey Lipinski's rules of five with zero or one violation, respectively. All tested compounds are expected to be a P-gp non-substrate. They are considered to be poorly absorbed by the intestine, except for compound IV. They are able to cross BBB with a probability ranging from 0.7 to 0.9. Most compounds exhibited limited permeability as indicated by logP values in the range of 4.25-7.59, except for compound IV. Concerning CYP inhibition, all compounds were predicted as inhibitors with a probability higher than 0.5, as shown in Table 7. The results predict that compounds IV and Va-i could exhibit acceptable pharmacokinetic and ADME properties (Tables 7 and 8). -------------  A mixture of 2-phenylindole (I) (0.53 g, 1.55 mmol, 1 eq), β-nitrostyrene (II) (0.25 g, 1.70 mmol, 1.1 eq), and sulfamic acid (0.02 g, 0.31 mmol, 0.2 eq) in methanol (30 mL) was heated at reflux for 12 h. After removing the solvent in vacuo, the residue was extracted with EtOAc, washed with saturated NaHCO 3 solution and brine, dried over MgSO 4 , and evaporated under reduced pressure to give a crude product, which was subsequently purified via flash chromatography on silica gel using EtOAc/hexanes (1:4) to obtain the desired product as an oil. This oil was solidified by being dissolved in DCM, subjected to the addition of hexanes, and left overnight.

EGFR Inhibitory Assay
The most potent antiproliferative derivatives (Va, Ve, Vf, Vg, and Vh) were also tested for EGFR inhibitory activity as a potential target for their antiproliferative activity [59,66]. See Supplementary Materials.

BRAF V600E Inhibitory Assay
Compounds Va, Ve, Vf, Vg, and Vh were further tested for BRAF V600E inhibitory activity, and the results are shown in Table 2 as IC 50 values [31,67]. See Supplementary Materials.

VEGFR-2 Inhibitory Assay
The inhibitory activity of compounds Va, Ve, Vf, Vg, and Vh against VEGFR-2 was determined utilizing kinase-glo-luminescent kinase assays with sorafenib as the reference drug [63,72]. See Supplementary Materials.

Caspase-3 Assay
The most effective derivatives, compounds Va, Ve, Vg, and Vh, were evaluated as caspase-3 activators against a human epithelial cancer cell line (A-549) [73]. See Supplementary Materials.

Caspase-8, Bax, and Bcl-2 Level Assays
Compounds Va and Vg were explored further for their impact on caspase-8, Bax, and Bacl-2 levels against a human epithelial cancer cell line (A-549) using doxorubicin as a reference [47]. See Supplementary Materials.

Conclusions
In this study, we presented the design, synthesis, and antiproliferative and apoptotic activities of a few novel indole-based derivatives (IV and Va-i). Various spectroscopic methods of analysis were used to confirm the structures of compounds IV and Va-i. Compounds IV and Va-i exhibited no cytotoxic effects in the cell viability test, and the cell viability for the compounds tested at 50 µM was greater than 89%. Compounds IV and Va-i demonstrated promising antiproliferative activity, with GI 50 values ranging from 26 nM to 104 nM against the cancer cell lines tested compared to erlotinib's 33 nM. The most potent antiproliferative derivatives Va, Ve, Vf, Vg, and Vh were tested for EGFR, BRAF V600E , and VEGFR-2 inhibitory activities as potential targets for their antiproliferative activity. Computational simulations confirmed the significance of the 5-chloroindole moiety in improving the fitting of the compound within the active sites of EGFR, Braf V600E , and VEGFR-2, highlighting the effect of the substituent at the third position of the indole scaffold on the binding of compound. Moreover, the amide linkage at the second position of the indole is significantly involved in H-bond interactions within the VEGFR-2 active site. In addition, the 2-phenyl indole scaffold bound considerably within the hydrophobic pocket of the binding sites. Our results showed good binding modes for compounds Va, Vg, and Vh within EGFR and Braf V600E . Also, docking results showed comparable fitting for compounds Ve and Vg within the active site of VEGFR-2. In silico ADME and pharmacokinetic prediction validated that the compounds have acceptable bioavailability and pharmacokinetic profiles. These findings revealed that compounds Va, Ve, Vg, and Vh displayed substantial antiproliferative effects and that they may operate as multi-kinase inhibitors, making them viable lead compounds for additional structural modifications.